The norepinephrine transporter (NET) is responsible for limiting the actions of norepinephrine (NE) in the brain and in the sympathetic nervous system through rapid reuptake of released NE. This proposal attempts to identify mutations in the human NET (hNET) gene that may contribute to cardiovascular and psychiatric disease. DNA samples from patients with orthostatic intolerance and depression will be genotyped for hNET mutations. Genomic DNA will be isolated from blood or tissue samples, and, using primers directed against hNET, polymerase chain reaction and sequence analysis will be performed to determine if polymorphisms exist in coding region of hNET. Mutations that result in amino acid substitutions will be examined for effects on hNET through site-directed mutagenesis of an expression vector containing hNET sequences, transfection and performance of [3H]NE transport assays and protein analysis through Western and radioligand binding assays. Known polymorphisms identified in hypertension will also be examined for their effects on hNET function. Through identification of hNET mutations in diseases affecting noradrenergic function, further understanding of the mechanism of noradrenergic neurotransmission will be achieved and successful therapies for these disorders can be developed that selectively target hNET.